This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The Vector Core directs the production of the oncoretrovirus, lentivirus, and recombinant AAV vectors. The virus vector production facility maximizes resource utilization and efficiency by centralizing the production of recombinant virus vector production, such that it is all done using standard protocols and the same strict guidelines for all of the gene therapy projects that are pursued at TNPRC. By providing vector core services, the VDPC will have a large impact not only on the Gene Therapy Program, but on other divisions such as Comparative Pathology, Microbiology and Immunology within the TNPRC, but also Departments and Centers within the Tulane Health Sciences Center and Louisiana State University Health Sciences Center. In collaboration with the Division of Microbiology and Immunology, the VDPC has begun to generate vectors that express recombinant proteins with investigators at the TNPRC.Produce recombinant vector preparations for collaborative research projects. The core lab will generate a series of lentivirus and AAV vectors for use in the Division Krabbe's research program. The vectors that will be developed will all express GALC (the defective gene in GLD). Vectors will be developed that mediate constitutive gene expression regardless of the cell type transduced, and vectors that mediate oligodendroglial lineage specific expression through the incorporation of cell-type specific promoter elements will be created as well. These vectors will be used for direct injection studies in the twitcher mouse and rhesus Krabbe's models. Additionally, a wide range of new gene transfer-related techniques and procedures are routinely developed each year. As investigators discuss their respective research projects, it is anticipated that new techniques will be developed, and that additional services will become available in the future. Any new techniques developed through our research efforts including our work with stem and progenitor cell populations and molecular assays will be made available to investigators.Develop isolation techniques for the purification of alternative serotypes of AAV. Nine novel AAV serotypes have been isolated from a number of species, including nonhuman primates (AAV7, AAV8 and AAV10) plus numerous additional novel serotypes are currently being isolated. These serotypes primarily differ from one another by their capsid proteins and all but AAV6 have been determined to be unique serotypes based on antibody cross reactivity studies. AAV6 appears to be a recombinant between AAV1 and 2. The specific serotypes of AAV differ in the amino acid content of their capsid proteins and these differences that have been demonstrated to result in variable cellular tropism in murine tissues such as the liver, skeletal muscle and brain. The differences in gene transfer observed for each serotype likely result from differences in the utilization of cellular receptors as evidenced by the ability of AAV2 to utilize heparin sulfate as a receptor, while other AAV serotypes do not. The receptor moiety for AAV5 has been identified as the platelet-derived growth factor receptor (PDGFR). Recently, the 37/67-kilodalton laminin receptor has been shown to act as a receptor for AAV serotypes 2, 3, 8 and 9, while the receptor for the remainder of AAV serotypes remain unknown. AAV vectors are then purified by two different methods depending on the serotype of the vector. AAV2 vector is purified by the single-step gravity-flow column purification method based on its affinity for heparin. The core will develop a multistep CsCl gradient centrifugation protocol to effectively isolate the alternative serotype vectors.Develop effective quality control assays for LV and AAV vectors. When requested, the VPC will conduct assays to determine if AAV or LV vector preparations are sterile and free of endotoxin contamination. These assays will be put into place during the next funding cycle. Sterility tests will be conducted by incubating an aliquot of the vector in LB medium at 37oC for 48 hours and transferring the medium to LB agar for an additional 24 hours. Results of the assay are then reported as number of colonies per ml of supernatant. To determine the amount of endotoxin present in a vector preparation, the VPC proposes to use the Quantitative Chromogenic LAL (limulus amebocyte lysate) Assay (BioWhittaker Inc., Walkersville, MD). The assay is based on the principle that gram-negative bacterial endotoxin catalyzes the activation of a proenzyme in the limulus amebocyte lysate which, in turn, causes the release of a colored compound from the substrate. The amount of endotoxin can be detected chromogenically with the correlation between absorbance and endotoxin concentration being linear in the 0.1 to 1.0 EU (endotoxin units)/ml range.Vector dissemination and biodistribution analysis. The VPC will establish the tools required to aide investigators in the study of vector dissemination and biodistribution in vivo, which is an important and informative aspect of viral gene transfer experiments. For these experiments, The VPC will use TaqMan real time PCR technology. Primer/probe stocks will be created to target promoter (e.g.- CMV, TBG, etc), transgene (e.g. EGFP, GALC, etc) and poly A (SV40, BGH, etc), respectively. These primer sets will be used to assay total cellular DNAs prepared from mononuclear cells and/or tissue samples. The genomic DNA will be subjected to highly sensitive real time PCR using an appropriate set of primers and probe to amplify vector sequences and quantify vector genomes present in the tissues analyzed. TaqMan-based biodistribution analysis will be validated for each set of primer/probe and target sequences. The samples will be analyzed using the equipment available in the Pathogen Detection and Quantification Core in the Division of Microbiology. Briefly, known copies of the vector genome to be analyzed will be spiked into 100 ng of monkey or mouse cellular DNA for TaqMan analysis. The output information of the spiked samples are informative in terms of the sensitivity of detection and possible interference of cellular sequences.
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